Many widely known recombinant DNA techniques involve replicating or amplifying DNA. One such example is the cloning of an insert DNA into a target DNA fragment. During this procedure, the target fragment is typically digested with a restriction enzyme such as EcoRI. Similarly, the insert DNA, having the gene of interest, is digested with the same enzyme. In one type of restriction enzyme digestion, cleavage of both the target DNA and insert DNA leaves overlapping 3′ or 5′ nucleotide fragments on each end. These cohesive, overlapping fragments or “sticky ends” are well-known properties of some restriction enzymes. Incubation of the target and insert DNA together at an appropriate temperature allows the insert DNA to noncovalently bind to the target DNA. The target DNA and insert DNA are held together by hydrogen bonding of the cohesive ends. Further incubation with an enzyme, such as DNA ligase, results in ligation of the insert DNA to the target nucleotide strand.
Another method of adding an insert nucleotide fragment into a target DNA is known as blunt-end ligation. Digestion with some restriction enzymes, such as SrfI (GCCC/GGGC), SmaI (CCC/GGG), or Eco RV (GAT/ATC) do not leave any 3′ or 5′ overhanging nucleotides at the enzyme splice site. These enzymes are known as “blunt-end” enzymes due to this feature of their enzymatic activity. After digestion, blunt-end restriction enzymes maintain single 5′ “terminal” phosphates on both sides of the restriction site. These terminal 5′ phosphates are required by DNA ligase for any subsequent religation of the digested DNA sequence.
The ExoClone™-PCR Cloning Kit (Sigma, St. Louis, Mo.) utilizes a method by which multibase sticky end ligations between PCR products and suitably cleaved plasmid DNA is accomplished. Amplified DNA inserts are produced using specially designed primers whose 5′ ends are cohesive with EcoRI cleaved vectors, i.e., 5′ pAATTC. A modified mix containing thiodeoxyguanosine triphosphate (sdGTP) and deoxyguanosine triphosphate (dGTP) is used to amplify high GC and long (up to 4 kb) targets. Because exonuclease III cleaves phosphorothioates extremely slowly, if at all, digestion with exonuclease III exposes the bases of the 5′ termini which are cohesive with the EcoRI digested vectors. This method is limited to cloning DNA into EcoRI cleaved vectors because the EcoRI recognition site is the only commonly used restriction site whose 5′ four base overhang is punctuated by a base, i.e., guanine, not represented within the 5′ pAATTC overhang.
Several methods have been devised for preferentially cloning insert DNA fragments into target sequences in one orientation. These methods are commonly known as directional cloning techniques and have been devised to position genes in the correct 5→3′ orientation. Directional cloning is commonly performed by digesting the target nucleotide sequences with two different restriction enzymes. This method results in a molecule with dissimilar DNA ends at the target insertion site. The insert DNA is then digested with the same two restriction enzymes thereby having two dissimilar DNA ends that correspond to a specific orientation in the target insertion site. By following this procedure, the insert DNA only binds to the target sequence in one orientation.
Another method of directionally cloning an insert into a target sequence uses an exonuclease, such as Exonuclease III, to create the “sticky ends”. See Kaluz, et al., Nucleic Acids Research, 1992, 20:4369-4370; U.S. Pat. Nos. 5,580,759, 5,518,901, 5,688,669 and 5,744,306. In this method, insert DNA fragments are digested with exonuclease III, a double strand specific exonuclease that catalyzes the stepwise release of nucleotides from the 3′ hydroxyl termini of double stranded DNA, to produce cohesive ends. Digestion with exonuclease III is performed at low temperatures for very short times, usually 30-90 seconds, in order to prevent excessive degradation. After a timed digestion, the insert fragments have 5′ overlapping nucleotide tails. These 5′ nucleotide tails are engineered so that the 5′ ends hybridize in one orientation upon base pairing to the target plasmid DNA molecule thereby resulting in a relatively simple method of directional cloning. While the use of exonuclease III provides a relatively simple method for directional cloning, it may be difficult to control the length of the generated cohesive ends which may be critical when cloning small size insert DNA.
There are several drawbacks, however, with single restriction enzyme digestion cloning methods and double restriction enzyme digestion directional cloning methods. For instance, digesting both the target DNA and insert DNA with restriction enzymes can be time consuming and multiple enzyme digestions increase the risk that either the target or insert DNA sequence will be cleaved at an internal restriction site. It is preferable that the sequence of the target DNA be known so that restriction enzymes are not selected which would cleave the sequence at an internal restriction site. Additionally, some restriction enzymes cleave very poorly, or not at all, when their recognition sequence is at or near the termini of a DNA strand.
Accordingly, it is desirable to formulate reagents and components for use in cloning and other recombinant DNA methodologies which could be utilized independent of the sequence being replicated or amplified. Such formulations would obviate the need for tedious control of restriction digestion and at the same time have minimal impact on PCR amplification performance. The provision of such process and reagent mixtures would avoid tedious or expensive aspects of current directional cloning such as multiple restriction enzyme digestion, addition of extra nucleotides to the insert and/or the use of multiple primers or linkers.